Ultra-high Yield Intravenous Immune Globulin Preparation

ABSTRACT

An efficacious large-scale alcohol-free plasma fractionation production process which produces a high-yielding, non-denatured, double viral-inactivated intravenous human immune gamma globulin (IgG) product. The process employs one or more salts from a group of salts comprising sodium citrate, sodium acetate, sodium gluconate, ammonium sulfate, sodium chloride, sodium sulfate and ammonium chloride in two initial fractionation steps, followed by diafiltration to remove those salts employed. A process which employs alcohol via the process of the disclosed inventive method is also disclosed.

This Application for Patent is a Divisional of U.S. patent applicationSer. No. 11/358,431, filed Feb. 21, 2006, which is aContinuation-in-Part of U.S. patent application Ser. No. 11/232,527,filed Sep. 22, 2005, which is a Continuation-in-Part of U.S. patentapplication Ser. No. 11/217,956, filed Sep. 1, 2005.

FIELD OF INVENTION

This invention relates generally to methods for immune serum globulinpurification, and, more particularly, to methods for alcohol-freeseparation of immune globulin from blood plasma or other blood basedmaterial. Interestingly, the method of the instant invention also may beemployed using alcohol.

BACKGROUND AND DESCRIPTION OF RELATED ART

Commonly, contemporary methods for separation of immune globulins (IgG)from blood plasma or other blood based material depend upon early workby Edwin J. Cohn. As found in U.S. Pat. No. 5,177,194 issued Jan. 5,1993 to Maria E. Samo, et al. (SARNO), “One scheme in widespread use isthe well-known Cohn fractionation method, which is based on differentialprecipitation using cold ethanol.” Cohn et al. J. Am. Chem. Soc. 68, 459(1946).

A U.S. Pat. No. 2,390,074 issued Dec. 4, 1945 to Edwin J. Cohn (Cohn)disclosed use of alcohol, acetone and dioxane as precipitants in suchfractionation processes. Continued dependence upon alcohol as aprecipitant is further demonstrated in U.S. Pat. No. 6,893,639 B2 issuedMay 17, 2005 to Joshua Levy, et al. (Levy), wherein it is stated, “Theconventional industrial methods of immune globulins purification fromblood plasma are based on cold ethanol fractionation whichco-precipitate groups of proteins based on their isoelectric points atgiven alcohol concentration at sub-zero temperatures.”

Cohn's work was stimulated by the need of the military for a stablesolution for use as a plasma volume expander during World War II toreplace lyophilized plasma. Consequently, the Cohn method focused onoptimizing the process for separating the albumin fraction whichprovides the osmolality necessary for plasma volume expansion.

Even so, the use of alcohol precipitants is not without difficulties, asillustrated by Cohn, “Some protein precipitants, such as alcohol, have atendency to denature many proteins with which they come in contact, thedanger of denaturation increasing with concentration of the alcohol andincrease in temperature. For many proteins, it has been found advisableto exercise considerable care in mixing the precipitant with the plasmaor other protein solution in order to avoid denaturation of theprotein.” For this reason, it is considered prudent to provide analcohol-free method for blood plasma and other blood based materialfractionation, including IgG purification.

Further considerations of combining ethanol and water may be warrantedrelative to denaturation of proteins. For example, if one adds 500 ml ofethanol (100%) to 500 ml of water, one does not obtain 1000 ml of 50%ethanol. Rather, the final volume is approximately 956 ml. It issurmised that the reduction in volume is due to a tight binding betweenthe ethanol and water molecules. Such binding may be a cause of changesin protein configuration resulting in some permanent denaturation ofprotein molecules which remains after ethanol is removed and water isreturned.

In the 1970's, chromatography was found to be useful in the separationand purification of plasma proteins. Chromatography separates plasmaproteins by specifically targeting unique characteristics of each,including molecular size (gel filtration), charge (ion exchangechromatography), and known interactions with specific molecules(affinity chromatography).

The use of various chromatographic methods on an industrial scale hasbeen adopted for the isolation of small-weight, high-value proteins,such as Factor VIII, from plasma, and for the final purification ofgamma globulin after separation from the plasma by Cohn, or modifiedCohn methodologies. However, chromatographic separation of thelarge-weight, lower-value fractions such as albumin and gamma globulin,on an industrial scale has not been found to be practical.

Two U.S. Patent Applications, filed by Edward Shanbrom, havingApplication Numbers 20030022149 (Shanbrom '149) and 20030129167(Shanbrom '167) filed Jan. 30, 2003 and Jul. 10, 2003, respectively,teach of use of carboxylic salts (e.g., trisodium citrate) as an agentfor enhancing formation of a cryoprecipitate from plasma. The method(s)of Shanbrom generally involve trisodium citrate and other citrate saltsas agents for enhancing production of blood clotting factors fromcryoprecipitate.

Shanbrom '149 teaches in paragraph 0009 that “It is an object of thepresent invention to provide enhanced yields of cryoprecipitate.”Shanbrom also teaches, in paragraph 0011, that carboxylic acids areeffective agents for enhancing the production of blood clotting factorsfrom the cryoprecipitate. Shanbrom '149 notes that the addition ofcitrate to plasma, especially at concentrations between two and tenpercent, by weight, does not appreciably denature labile proteins.Moreover, it is noted in Shanbrom '149 that citrate potentiates orenhances the killing of microorganisms by heat treatment.

Shanbrom '167 notes in paragraph 0015 that, “Not only does added citrateincrease the amount of cryoprecipitate, it simplifies the process bydecreasing the requirement for freezing . . . ” plasma in order toharvest cryoprecipitate. Shanbrom clearly teaches use of production of acryoprecipitate for the purpose of fractionating products from thecryoprecipitate through the use of trisodium citrate in concentrationsof two to ten percent.

While Shanbrom '149 and '167 deal directly with extracting labilecoagulation products from a cryoprecipitate formed through use ofcitrate compounds, particularly trisodium citrate, and with killingmicroorganisms in the cryoprecipitate using the citrate compounds, theinstant invention deals directly with extracting non-labile products(e.g., albumin, gamma globulin and alpha-1-antitrypsin) from asupernatant formed through use of salt compounds. Shanbrom neitherteaches nor addresses using a supernatant in any way.

In the 1950's, it was discovered that a “cryoprecipitate” derived fromblood-based material, contained various factors was useful in treatingclotting disorders such as hemophilia. Such a cryoprecipitate, as thename implies, was obtained by freezing blood plasma followed bycontrolled thawing at zero to four degrees Centigrade to form a liquidsuspension of the precipitate. A supernatant derived from thecryoprecipitating process was then available for fractionation usingmethods according to Cohn to produce albumin and gamma globulin.Subsequent developments led to fractionation of cryoprecipitate intopure concentrates of Factor VIII, von Willebrand Factor, and otherclotting factors. Such may be accomplished by using non-alcoholicseparations and chromatographic purification.

BRIEF SUMMARY AND OBJECTS OF THE INVENTION

In brief summary, this instant invention provides novel and effectivemethods of isolating gamma globulin from plasma and formulating it intoan intravenous injectable preparation. Accordingly, this invention,which may be defined to be “an ultra-high yield intravenous immuneglobulin preparation,” achieves higher yields of a superior qualitygamma globulin by directly and expeditiously separating the gammaglobulin from the plasma by means of a non-denaturing precipitant, whichmay be selectively chosen from a group of organic salts including sodiumcitrate, sodium acetate, and sodium gluconate and from a group ofinorganic salts including ammonium sulfate, sodium chloride, sodiumsulfate and ammonium chloride. Two surprising characteristics of use ofthese salts are that (1) fractionation depends upon employing aneffective weight percentage solution and (2) that these salts areeffective in such fractionation when used alone and when used in acombined mixture of two or more salts wherein the combination has thesame effective weight percentage.

Also, addition of these salts to protein in solution proves to be not asreactive to removal of water as addition of ethanol by previous methods.A rapid isolation using these salts, removal of the resulting fractionfollowed by removal of salt from the resulting fraction and a quickrestoration of the internal water molecule of the protein has provedsuperior to ethanol fractionation using currently employed methods.

The inventive process is for fractionating blood-based products toproduce a useful, nondenatured immunoglobulin (sometimes referred to asIgG) product which involves the following critical steps:

(a) adding a sufficient first measure of a chosen salt or combination ofsalts to a quantity of blood-based material to be fractionated to bringthe added first chosen salt or combination of salts to a firstpredetermined concentration level which forms a supernatant product,free of euglobulins, the supernatant being separable from a residualpaste and separating the two;

(b) performing a second fractionation step by adding a second measure ofthe same or another chosen salt or combination of chosen salts to theseparated supernatant product to bring the chosen salt or saltscombination to a second predetermined concentration level which,thereby, forms a second separable product which is a paste and a secondresidual product which is a supernatant, the products thereafter beingseparated;

(c) forming a liquid dilution of the second separated paste product; and

(d) diafiltering the second separated and liquified paste product toform a low volume resulting product which is substantially free of thechosen salt or salts and ready for processing by currently known andpracticed procedures to complete production of the useful, non-denaturedIgG.

In step (a), the first volume utilizes an added concentrated saltsolution or addition of dry salt to the blood-based material to yield aneleven to thirteen percent solution by weight when mixed into a quantityof blood-based material. At this concentration, the added saltselectively dehydrates portions of the blood-based material to form asupernatant and a precipitated residual paste. Note that the supernatanthas a resulting salt concentration in the range of eleven to thirteenpercent concentration by weight of the selected salt or combination ofsalts. Preferably the concentration should approximate twelve percent.If desired, the residual paste may be further fractionated into bloodfactors including VIII, IX, von Willebrand and fibrinogen. Separation ofthe products may be accomplished by centrifuging or existing methodswhich are well known in chemistry art. The supernatant is retained forfurther processing.

In step (b), the second volume utilizes addition of more concentratedsalt solution or addition of a sufficient amount of dry salt to theretained supernatant to selectively dehydrate portions thereof to yielda second paste product and a residual second supernatant product. Thetotal concentration by weight of both the second paste and secondsupernatant should be in a range of approximately twenty-one totwenty-three percent. It is preferred that concentration by weight ofthe selected salt or combination of salts should approximate twenty-twopercent. As with step (a), if desired, the residual (in this case thesecond supernatant) may be further processed into a group of componentscomprising albumin, alpha-1-antitrypsin and other proteins. The productsmay be separated by centrifuging, filtering or other methods which arewell-known in the chemistry art.

Surprisingly, precipitation using salts does not appear to be dependentupon some sort of molar reaction. Rather, precipitation appears to bebased upon a simple percentage by weight relationship in both steps (a)and (b). In the following table (Table I), effective concentrations ofboth organic and inorganic salts are found. Note the effectiveconcentration by weight is the substantially the same percentage foreach salt listed.

TABLE I Examples of effective concentration of salts which may be used mSteps (a) and (b). Molar Weight in Molar Weight in a 12% a 22% MolecularSolution Solution Weight (for step (a)) (for step (b)) Organic SaltsSodium citrate 294 0.408 M 0.748 M  Sodium acetate 82  1.46 M 2.68 MSodium gluconate 218 0.427 M 0.783 M  Inorganic Salts Ammonium sulfate132 0.908 M 1.66 M Sodium chloride 58.5  2.05 M 3.76 M Sodium sulfate142 0.845 M 1.54 M Ammonium chloride 53.5  2.24 M 4.11 MAlso, surprisingly, the above listed salts may be used in anycombination if the total concentration by weight is maintained as citedfor steps (a) and (b).

In step (c), it is preferred to dilute the paste product with waterhaving approximately four times the weight of the paste product,although other volumes of water may be judiciously selected within thescope of the invention.

In step (d) a diafiltration system with a 30 KD filtering membrane maybe used to separate the selected salt or salts and excess water from theresulting product to permit further processing on an industrial scale.Note, that such filtering is made facile and possible by extractingeuglobulins from the supernatant in step (a). As used herein,euglobulins are defined to be those globulins which are insoluble inwater, but are soluble in saline solutions. Most importantly, ifeuglobulins are not removed from a solution and if the ionic strength ofthat solution is lowered towards deionized water (e.g., in the case ofthe instant invention), euglobulins foul a diafiltration system, therebyrendering it unuseable.

It is well-known that sodium citrate, has long been used in lowconcentrations during the collection, preservation and storage of bloodplasma. Subsequent diafiltration after use of high concentrations ofsodium citrate and/or other salts as a precipitant substantially reducesthe ionic strength and volume of the gamma globulin solution, permittingthe achievement of chromatographic purification on an industrial scale.

Following separation of gamma globulin from plasma by this method,albumin and alpha-1-antitrypsin are subsequently removed from theremaining proteins by methods available from Cohn or others. Theprocess, according to the instant invention, enables the separation ofgamma globulin without exposing it to the denaturing effects of ethanolused in the Cohn process, hence leaving the gamma globulin in a nativestate. The denaturing effects of alcohol include the formation ofpolymers, aggregates and fragments of the gamma globulin molecule.However, the use of single or combinations of earlier named selectedsalts stabilizes the plasma while bringing about precipitation ofsubstantially all of the coagulation proteins, thus preventing thegeneration of enzyme activators and proteolytic enzymes.

The absence of the denaturing effects of ethanol, the stabilization ofthe plasma with the selected salts, and the subsequent removal ofcoagulation proteins by means of the selected salts results in a gammaglobulin preparation which has very low anti-complementary activity.

In summary, the process of the instant invention employs highconcentrations of one or more preselected salts combined with subsequentremoval of those salts from the gamma globulin concentrate by means ofdiafiltration, a technique which became practical on an industrial scalein the 1980's. Final purification of the resulting gamma globulin isthen practically and effectively achieved through the use ofwell-established chromatographic purification techniques. The inventionreduces production costs as a result of higher yields, fewerfractionation steps, shorter overall processing time, lower energycosts, and lower chemical costs. Capital costs are less because ofreduced space requirements, reduced work-in-process, reduced processingtime, and elimination of the explosion-proof environments required forethanol processing.

Surprisingly, the method of the instant invention may employ alcohol inconcentrations similar to concentrations of salts mentioned supra. Whileconcerns of denaturization exists with the use of ethanol, results ofexperiments were interesting. A basic difference between historicalethanol procedures is quick removal of ethanol after completion of theprocedure according to method of the current invention.

Accordingly, it is a primary object to provide an effective intravenousgamma globulin preparation at a cost which is reduced from methods incurrent practice.

It is therefore a principle object to provide an alcohol-free method forpreparing gamma globulin.

It is an important object to provide such a method and preparation whichis high-yielding.

It is a further object to provide a gamma globulin preparation which canbe rapidly infused with greater patient tolerance than gamma globulinproduced by traditional methods employing alcohol.

It is an object to provide a previously unused method employing alcoholwhich produces a high yield and lower denaturization than contemporaryprocedures.

It is an object to produce gamma globulin having reduced in-processformation of polymers, aggregates, fragments, enzyme activators andproteolytic enzymes compared with similar preparations produced usingtraditional alcohol-based methods.

It is a further object to derive a cryoprecipitate as an optional methodaccording to the instant invention to form a liquid suspension of acryoprecipitate from which, through fractionation, Factor VIII, vonWillebrand Factor, and other clotting factors are produced.

These and other objects and features of the present invention will beapparent from the detailed description taken with reference toaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of a critical set of initial steps associatedwith the process according to the instant invention.

FIG. 2 is a flow diagram disclosing a series of steps which immediatelyfollow the steps seen in FIG. 1.

FIG. 3 is a flow diagram disclosing those procedural steps whichimmediately follow the steps seen in FIG. 2.

FIG. 4 is a flow diagram disclosing steps which immediately follow thesteps seen in FIG. 3 to provide a useful product.

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

Reference is now made to flow path elements illustrated in FIGS. 1-4.Generally, each rectangular box is used to illustrate a procedural step;each diamond is used to demonstrate a separation step; each ellipticalcylinder designates a product resulting from a preceding procedural orseparation step; and each circle is used to identify either a startingpoint or an off sheet continuation path point.

Reference is now made to FIG. 1 wherein an initial portion 10-1 of anpreferred IgG process flow path, generally numbered 10, is seen. Asindicated after initial starting point 20, a volume of plasma 30 to beprocessed is selected for processing. It should be noted that whileplasma 30 is used by example in this description of an illustratedembodiment, other blood-based products may be processed within the scopeof the instant invention. Also, after preparation for use, a separationstep 42 is used to separate a paste 44 from prepared cryo-poor plasma50, as is disclosed in more detail hereafter.

As part of procedure 40, selected frozen plasma 30 is warmed toapproximately five degrees Centigrade to form a prepared plasma. Whilefive degrees is the target plasma 30 process temperature, which shouldbe maintained throughout the following steps in process 10, atemperature range between limits of two to eight degrees may be heldwithin the scope of the instant invention. Plasma 30 may be useddirectly if not selected in a frozen state (e.g., thawed during theprocess of removing a cryogenic precipitate by customary methods).

A quantity of a salt or salts (which may be selected from salts listedin Table I, provided supra) is selected for addition to plasma 50 perprocedure 52. Due to the fact that a single salt or a combination ofsalts may be selected and used, it is prudent to consider an added saltquantity as “at least one salt,” as a quantity used may contain a singlesalt or a combination of salts in each of steps (a) and (b), recitedsupra. Generally, a salt or combination of salts maybe prepared insolution, added as dry salt or added as a combination of hydrated anddry salts (procedure 54). In any event, it is most important to bringthe total concentration of added salts to a predetermined concentrationby weight.

As an example, when sodium citrate is selected and used, a fifty percentsodium citrate solution is prepared by stirring five hundred grams ofsodium citrate into six hundred milliliters of purified water. Stirringtime should be thirty to sixty minutes or, alternately, until the sodiumcitrate is dissolved. At this point, dilute the mixture with pure waterto one thousand milliliters. Add 50% citric acid solution to the mixtureuntil a pH of 7.0 is reached.

As is well known in organic chemistry art, the following steps can beused to produce a fifty percent citric acid solution. Add 50 gm ofcitric acid to 60 ml of purified water. Stirring time should be about 30minutes or until the citric acid is in solution. After the citric acidis in solution, add enough purified water to bring the volume to 100 mland mix well. A portion of this solution, added to the 1000 ml of sodiumcitrate, adjusts the pH to 7.0. Therefore, add the citric acid to thesodium citrate solution until the pH of 7.0 is reached. It should benoted that a very little citric acid needs to be added to adjust to a pHof 7.0.

Preparatory to performing the first fractionation step (procedure 54 forsodium citrate), a volume of fractionation solution to be added toplasma 50 is calculated. It is a goal that the salt concentration (inthis case sodium citrate fractionation solution) should be twelvepercent by weight. Also the pH of the fractionation solution should beapproximately 7.0.

The formula, (Formula I) for calculating respective volumes offractionation solution (sodium citrate) and plasma 50 are as follows:

x=(C*V)/(0.5−C)

-   -   where: x is desired volume of 50% sodium citrate solution; C is        a desired fractional concentration by weight of sodium citrate;        (e.g. 0.12 or twelve percent): and V is volume of solution to be        diluted (e.g., volume of plasma 50).        An example of a calculation by Formula I is:

For a volume (V_(p)) of plasma 50 of 500 liters, and the desiredfractional concentration by weight of sodium citrate is twelve percent:

x=(0.12*500)/(0.5−0.12)=158 liters

-   -   Solving Formula I for C yields Formula II into which values of        volumes of plasma 50 and sodium citrate may be inserted as        follows:

C=(0.500*158)/(500+158)=0.12

For procedure 54, over a period of approximately five minutes, add theprepared sodium citrate fractionation solution (which may be at roomtemperature, i.e. approximately twenty degrees Centigrade) to plasma 50,which has a starting temperature of five degrees Centigrade. Gently stirwhile adding the sodium citrate solution. Once the sodium citratesolution is completely added to plasma 50, continue gently stirring theresulting slurry for approximately sixty minutes while reducing theslurry temperature to within a range of two to eight degrees Centigrade.(The slurry should maintain pH at approximately 7.0 to 7.1.)

Upon completion of procedure 54, centrifuge as procedure 56. It isrecommended that a flow-through centrifuge (e.g., a WestphaliaCentrifuge) be used to separate component parts of the slurry into asupernatant liquid 60 and a paste 62 by normal procedures for thoseskilled in the art, while maintaining temperature of the slurry in therange of two to eight degrees Centigrade.

While supernatant liquid 60, which contains virtually all of the IgG ofthe original plasma, is retained for further processing as an integralpart of the instant inventive method, paste 62 may be further processedto recover blood factors, including Factors VIII, IX, von Willebrand andfibrinogen.

For the second fractionation phase using sodium citrate, perform processstep 64 which adds additional sodium citrate fractionation solution tosupernatant liquid 60. Enough fifty percent sodium citrate is added toliquid 60 to increase concentration by weight of sodium citrate fromtwelve percent to twenty-two percent. Note that, for other salt or saltsused in step 64, the total concentration by weight of the at least onesalt used should be in a range of twenty-one to twenty-three percent,preferably in the range of twenty-two percent.

To calculate the volume of fifty percent sodium citrate to be added,Formula III is provided as follows:

C _(e)=((V ₆₀*C₆₀)+(V _(x) *C _(0.50)))/(V ₆₀ +V _(x))

-   -   Where C_(e) is the desired end concentration by weight of sodium        citrate; V₆₀ is the volume of supernatant liquid 60; C₆₀ is        sodium citrate concentration in supernatant liquid 60; V_(x) is        volume of fifty percent sodium citrate to be added, and C₅₀ is        concentration of fifty percent sodium citrate (ie. 0.5).

Note that the desired end concentration by weight of sodium citrate insolution is 0.22 or twenty-two percent.

Solving for V_(x) yields Formula IV which may be used to calculatevolume of sodium citrate to be added.:

V _(x) =V ₆₀*(C _(e) −C ₆₀)/(C _(0.50) −C _(e))

-   -   As an example, for a volume of V₆₀ of 552 liters; a        concentration by weight of C_(e) of 0.22; a concentration by        weight of 0.12 for C₆₀; and a concentration by weight of 0.50        for C_(0.05:)

V_(x)=197 liters

After adding volume V_(x) of sodium citrate, stir for two to four hours(though one to two hours is often sufficient for smaller volumes) whileretaining the temperature of this mixture between two and eight degreesCentigrade. Note that this solution will change color to a pale yellowas the additional sodium citrate is added and the mixture is stirred.

After stirring, per step 66, centrifuge the mixture, use a continuousflow centrifuge while maintaining the temperature in the range of two toeight degrees Centigrade to separate paste 70 from supernatant 72. Theresultant supernatant (supernatant 72) contains essentially no IgG.Virtually all of the IgG of plasma 50 is now found in paste 70.

Reference is now made to FIG. 2 as point 80 continues to point 80′ forflow path portion 10-2 of flow path 10. Contents of paste 70 includesIgG, other serum proteins and sodium citrate. The sodium citrate (and/orother used salt or salts) must be removed from paste 70 to permit IgG tobe isolated by ion exchange chromatography. First, paste 70 is liquifiedusing purified water (of about four times the volume of paste 70) asstep 90. Product of step 90 is an IgG rich solution 100. Initialconductivity of solution 100 is approximately 20 milliSiemens/centimeter(mS/cm).

Removal of sodium citrate (and/or other used salt or salts) isaccomplished by continuous diafiltration using purified water as asolvent in step 102 which separates solution 100 into removed sodiumcitrate 110 and desalted IgG retentate 112. Note that this step shouldbe consistently performed, independent of the selected at least onesalt. Completion of step 102 is indicated when the conductivity ofretentate 112 is reduced to 100-900 microSiemens/centimeter 16 (μS/cm).For diafiltration in step 102, a Millipore (or comparable) diafiltrationsystem equipped with 30 KD cut-off membranes may be employed.

Viral inactivation of IgG rich retentate 112, associated with step 120,may be accomplished as a double viral inactivation step involving afirst solvent/detergent (S/D) method, followed by an augmented S/Dmethod. The first method employs raising the temperature of retentate112 to approximately twenty-seven degrees Centigrade (temperature mayrange from twenty-four to thirty degrees Centigrade). A sufficientvolume of Triton X-100 or Tween 80 is then added to make a one percentsolution and sufficient Tri-N-Butyl Phosphate to make a three tenths ofone percent solution to make a first S/D added mixture. The first methodcontinues by incubating the first S/D added mixture at twenty-sevendegrees Centigrade for three hours during which time lipid envelopedviruses are inactivated. From this point, procedures currentlyavailable, inactivation and fractionation processes may be employed.However, a currently preferred process is hereafter provided forcompleteness.

For step 120, a S/D concentrate may be made as follows:

-   -   Add 30 milliliters of Tri-N-Butyl Phosphate to 800 milliliters        of purified water. Mix well. Add 100 milliliters of either        Triton X-100 or Tween 80 to the mixed solution. Again, mix well        to provide a final, mixed S/D solution. Add enough purified        water to bring the total volume of the final mixed solution to        1000 milliliters. One more time, mix well. So made, the final        solution is a 10× concentrate. Add 100 milliliters of this        concentrate to each 900 milliliters of retentate 112 to form the        first S/D added mixture.

After three hours of incubation, add, to the solution resulting from thefirst S/D method, sufficient formaldehyde to make a three tenths of onepercent solution and sufficient phenol to make a three tenths of onepercent solution to form an augmented mixture to begin the augmentedmethod phase of step 120. Incubate at approximately twenty-seven degreesfor an additional three hours, after which time non-enveloped andenveloped viruses are inactivated.

For step 120, an “augmented” concentrate may be made as follows:

-   -   Add 13.4 milliliters of thirty-seven and one-half percent        formaldehyde solution to 900 milliliters of purified water. Mix        well. Add fifty grams of phenol (reagent grade) to this mixture.        Again, mix well. Add enough purified water to bring the total        volume of the “augmented” preparation to one thousand        milliliters. Once more, mix well. This preparations contains        50,000 parts per million each of formaldehyde and phenol (five        percent of each). Measure the volume of the first S/D added        mixture. Add 167 milliliters of augmented concentrate to each        833 milliliters of first S/D added mixture to form the augmented        mixture.

Step 120 is completed by cooling the processed augmented mixture to atemperature of two to eight degrees Centigrade. So cooled, the augmentedmixture becomes IgG virus inactivated (VI) solution 122.

Alternatively, viruses may be removed by other methods (e.g.,chromatography, nanofiltration, pasteurization), if desired.

Step 124 involves use of column chromatography to remove viralinactivation chemicals. Such may be accomplished by the followingsub-steps:

-   -   1. Set up a short, wide column with Toyopearl CM-650C resin. The        Toyopearl resin is a weak cationic exchange resin used to        capture IgG in solution 122 while permitting other proteins from        solution 122 to flow through the column. It is important that        the conductivity of solution 122 be in a range of 100 to 900        microSeimens/centimeter (μS/cm). (It is preferable that such        conductivity is in a range of 400 to 600        micro-Seimens/centimeter.) IgG from plasma 50 binds to the        exchange resin in the low ionic strength solution.    -   2. Introduce solution 122 into the exchange resin at a slow        rate. Collect effluent liquid from the column and measure the        effluent liquid at 280 nano-meters in a one centimeter silica        cuvette in a high quality spectrophotometer. (As an example, a        Beckman DU-7 with a deuterium light source may be used.) It        should be noted that optical density of the effluent will        increase as proteins are introduced into the resin column.        Phenol in the viral inactivation solution (if used) also can        increase measured optical density. After all of solution 122 has        passed through the resin column and sterilants are washed from        the column, begin collecting the effluent when measured optical        density increases from its original value. A rise in optical        density is indicative of protein in the effluent. After a        period, optical density drops down to a level which is        indicative of little or no protein in solution. At this point,        collecting may cease. At this point, it is preferable to        thoroughly wash the resin with deionized water. Bound material        is IgG, identified along path 10-2 as bound IgG 130. Collected        effluent from the column includes all of the protein from plasma        50 except for IgG. Ibis effluent is effluent solution 132. It is        recommended that serum protein electrophoresis be performed on        effluent solution 132 to confirm that little or no IgG has been        released into solution 132.

Depending upon size of the resin column, prepare a volume of two percentsolution of sodium chloride. Application of the sodium chloride is usedto effect release of attached IgG from resin particles. As is well-knownin chemistry art, a two percent solution is made by mixing 20 grams ofsodium chloride into one liter of deionized water. Sufficient volume oftwo percent sodium chloride solution should be made to equal about tentimes the volume of the resin column.

For step 134, add the sodium chloride solution to the column, collectingeffluent from the column. Concurrently, measure optical density of theeffluent solution at 280 nanometers using a spectrophotometer with a onecentimeter silica cuvette. Resultant optical density (OD) will be foundto suddenly increase as IgG is uncoupled from the resin and deliveredinto the effluent. Collect all high OD measured solution. When the OD ofthe effluent drops to a lower (normal) range, cease collecting thesolution. Resulting solution is IgG solution 140. Note that a high OD isindicative of protein content in solution, and that solution 140 maycontain small amounts of IgM and IgA, which requires further removal. Inaddition solution 140 contains sodium chloride which must be removedbefore any pure IgG can be isolated.

Reference is now made to continuation point 150 in FIG. 2 whichcontinues to continuation point 150′ in FIG. 3 for flow path portion10-3 of flow path 10. Sodium chloride is preferably removed fromsolution 140 by continuous diafiltration employing a diafiltrationsystem. Such may consist of a Millipore (or comparable) diafiltrationsystem equipped with 30 KD cut-off membranes. As performed in step 152,the diafiltration solvent is purified water. As may be noted, initialconductivity of solution 140 is approximately fiftymilliSiemens/centimeter (mS/cm). At completion of diafiltration,conductivity is reduced to 100900 microSiemens/centimeter (μS/cm).

The products of diafiltration are an IgG rich retentate 160 and removedsodium chloride 164. It is recommended that serum electrophoresis beperformed at this step in the process to confirm protein fractions inretentate 160.

Step 166 is a final step for purifying IgG rich retentate 160. For step166, it is preferred to set up a short, wide resin column with ToyopearlQAE-550C resin. Such resin provides a strong anionic exchange forcapturing other proteins in IgG rich retentate 160, while permitting IgGin solution to flow through the column. It is important thatconductivity of retentate 160 be in a range of 100 to 900microSeimens/centimeter, and preferably, within a range of 400 to 600microSeimens/centimeter. In this manner, IgG in retentate 160 will passthrough the resin column in Step 166 without binding, while otherproteins, including IgM and IgA, will bind to resin in the column andthus be removed from solution. In this manner, any contaminatingresidual proteins 170 are effectively separated from a purified IgGsolution 172.

As the process is continuous, it is recommended that IgG solution 172 becollected and the OD measured at 280 nm. Collect the high OD effluentsolutions. When the measured OD drops, cease collecting. The pooledsolution is relatively dilute.

The pooled solution is concentrated using step 180 via ultrafiltration.For such ultrafiltration, a hollow fiber filter may be used, or aMillipore ultrafiltration system (Pellicon) or equivalent, (10K to 30Kdalton retentation) to concentrate to a twelve percent IgG solution 182.Excess water 184 is removed in the process of step 180. The resultingtwelve percent concentrate should have only a trace amount of sodiumchloride and the pH should be approximately seven. Conductivity shouldmeasure about 100 to 900 microSiemens/centimeter.

To stabilize the twelve percent IgG solution 182, add (step 190) amaltose or sorbitol solution to dilute the twelve percent solution toexactly ten percent. The final ten percent solution (IgG solution 192)should contain approximately five percent maltose or sorbitol (whicheveris used).

Optionally, to remove viruses 204 from IgG solution 192, nanofiltrationmay be performed by passing the ten percent solution 192 through a virusretaining membrane (step 200) to produce a nanofiltered concentrate 202.

Reference is now made to continuation point 210′ in FIG. 4, for flowpath portion 10-4 of flow path 10, which continues from continuationpoint 210 in FIG. 3. As is standard procedure, (depending upon executionof prior option step 200) either stabilized IgG concentrate 192 ornano-filtered IgG concentrate 202 is diluted with deionized water instep 220 to produce a bulk purified IgG solution 222. Contaminatingbacteria may be removed by passing solution 222 through a sterilizingfilter in step 230 to produce a sterilized bulk IgG solution 232.Removed contaminating bacteria 234 may be disposed of by methodscurrently known in the art.

Resulting sterile solution 232 may be filled into vials per standardprocedures in step 240 to produce a lot 242 of vials of solution 232. Asrequired for quality assurance, final testing and inspection of lot 242may be made in step 244 in cooperation with step 246 to produce a lot250 of validated vials of solution 232, with any discard 252 beingremoved therefrom.

Reference is now made to FIG. 1 wherein flow chart 10-1 discloses stepsfor separating out a paste from which other products, e.g. Factor VIII,von Willebrand Factor, and other clotting factors which may befractionated, are seen. Steps associated with product 44 seen in FIG. 1are optional and may be followed to produce a cryoprecipitate from whichFactor VIII, von Willebrand Factor, and other clotting factors can beremoved. In this case, procedure 40 is defined to gradually warm plasma30 to zero to four degrees Centigrade. Such warming results in a thawedplasma in which a cryoprecipitate is suspended. Per step 42, the thawedplasma is centrifuged to yield the cryoprecipitate in the form of apaste 44. Paste 44 may be subsequently separated and processed by knownmethods to provide Factor VIII, von Willebrand Factor, and otherclotting factors. The remaining separated material, named cryo-poorplasma 50, is processed as disclosed supra.

Results of a Fractionation Procedure Performed According to the InstantInvention:

In order to show the efficacy and precision of separation of steps ofthe instant invention disclosed herein, the following results, usingsodium citrate, have been extracted from a laboratory report, dated Aug.8, 2005.

In the procedure, fresh frozen human plasma was used. As is typical insuch procedures, a pool was made from four to eight bags of thawedplasma (see step 40, FIG. 1). Commercial equipment available fromBeckman-Coulter was used to evaluate various fractions as they becameavailable. The Beckman, “Appraise”, densitometer was used to scan theBeckman agarose gels for serum protein electrophoresis as part of aParagon Electrophoresis System. For each fraction made from the pool,between three and five gel slits were loaded with five microliters ofproduct. Results were averaged to obtain a better representative resultfor each fraction. Such results are found in tables provided hereafter.

The gels were electrophoresed for twenty-five minutes at 100 VDC at a pHof 8.6 and later stained with a Paragon blue stain. The Appraisedensitometer was used to scan the stain-dried gels at a wavelength of600 nanometers twice for each gel slit (ten gel slits per gel wereused). An average graphic representation of the distribution of fivedifferent protein fractions, based upon density of attached dye as wellas a numeric presentation of each fraction was derived. The numericpresentation was based upon a computer analysis of peaks and valleys ofgenerated graphs at selected locations within the gel pattern asoccurred between the anode and cathode on each gel. Presentation valueswere totaled and dye percentage was divided by the total dye amount toprovide a percentage for evaluation. Note that the grand total, summingeach individual blood fraction always equals one hundred percent.

As seen in Table II below, the five different protein fractions areidentified as: Albumin, Alpha1, Alpha 2, Beta and Gamma globulin. Beforefractionation, a sample was removed from the pool and electrophoresed todetermine the average fractional values of each fraction beforebeginning fractionation. Representative results for the average basepool material are listed below as percentages of whole plasma:

TABLE II Percentage content of each fraction Albumin Alpha 1 Alpha 2Beta Gamma Base plasma 61.2 7.1 9.7 13.0 8.9Plasma (i.e., plasma 50) from the pool was treated with the addition ofa volume of fifty percent sodium citrate to a volume of plasma to make atwelve percent solution of sodium citrate (step 54). This mixture wasstirred for sixty minutes at two to eight degrees Centigrade and wasthen centrifuged (Step 56) for sixty minutes at two to eight degreesCentigrade. The resulting supernatant solution 60 was measured. Theremaining paste 62 was weighed and put into solution by addition ofdeionized water.

The two solutions (60 and 62 (dissolved)) were electrophoresed usingprocedures cited supra, the results of which are summarized in TableIII, below:

TABLE III Resulting percentage concentrations Albumin Alpha 1 Alpha 2Beta Gamma 12% Paste 62 (dissolved) 67.8 27 9.1 20.4 nd* 12% Supernatant60 28.6 1.5 10.7 40.3 16.1 *nd = none detected

-   -   There was no gamma globulin found in Paste 62 (dissolved).        However, there was gamma globulin found in Supernatant 60.        Next, sufficient fifty percent solution sodium citrate was added        to supernatant 60 (step 64) to obtain a final mixture that        contained twenty-two percent sodium citrate. This solution was        also stirred for sixty minutes at two to eight degrees        Centigrade. After centrifuging (see step 66) for sixty minutes        at two to eight degrees Centigrade, the resulting supernatant        solution 72 was measured. The remaining paste 70 was weighed and        put into solution (step 90) by the addition of deionized water        (four times weight of paste 70 in milliliters) to form IgG rich        solution 100. Samples of supernatant 72 and IgG rich solution        100 were electrophoresed by the procedure cited supra, the        results of which are summarized in Table IV below:

TABLE IV Percentage concentrations of indicated solutions Albumin Alpha1 Alpha 2 Beta Gamma 22% Supernatant 72 82.4 13.9 3.6 nd* nd* 22% Sol.100 (dissolved) 16.7 1.3 10.5 32.5 39.0 *nd = none detectedThere was no gamma globulin found in supernatant 72. However, there wasgamma globulin found in IgG rich solution 100.

The 22% supernatant fluid (which contained mostly albumin) containedessentially no beta and gamma globulin (i.e., none of such that wasdetected). The twenty-two percent paste solution 100 contained the gammaglobulin of interest for further fractionating to produce intravenousgamma globulin for injection. Note also that, in step 90, time should beallowed for the paste to solvate prior to performing electrophoresis. Inthe experimental process, a plasma fraction between twelve percent andtwenty-two percent sodium citrate was selectively isolated out for usein this isolation procedure.

To remove sodium citrate (step 102) trapped in the twenty-two percentpaste solution, a Pellicon unit was selected to diafilter solution 100.On the average, about seven times the volume of solution 100 wasrequired to diafilter the sodium citrate and bring conductivity of theresulting solution down to a range between 400 and 800microSiemens/centimeter (μS/cm), before performing any column work.

After diafiltration, the desalted protein solution 112 was treatedelectrophoretically to determine any changes or losses as a result ofdiafiltration step 102. Because sodium citrate was removed, proteinmovement in the electrophoretic pattern was changed somewhat throughlack of interference with a contained salt. The resulting pattern wassomewhat longer than a high salt concentration pattern. This elongatedpattern allowed IgG to separate more readily from beta globulin with aresulting increase in measured percentage as seen in Table V, providedbelow:

TABLE V Percentage content of diafiltrate fractions Albumin Alpha 1Alpha 2 Beta IgG Solution 112 16.6 1.5 9.9 26.9 45.2As seen in Table V, approximately forty-five percent of solution 112 wasgamma globulin and solution 112 exhibited better separation in theelectrophoresis pattern. Note, that the beta fraction went down withbetter separation in the electrophoresis pattern.

At this point, various currently employed methods could have been usedto purify the gamma globulin in solution 112. For that reason, thecompletion of this experiment could have varied from steps seen in FIGS.2-4. In the case of this experiment, the solution was first treated witha Solvent/Detergent solution of three hours at twenty-seven degreesCentigrade. Then an augmented sterilization solution, performedaccording to U.S Pat. No. 6,881,573, titled AUGMENTED SOLVENT/DETERGENTMETHOD FOR INACTIVATING ENVELOPED AND NON-ENVELOPED VIRUSES, issued toAllan L. Louderback, filed Sep. 12, 2003, was added to the mixture andfurther incubated for an additional three hours at twenty-seven degreesCentigrade. This dual inactivation treatment of the dialyzed diafiteredsolution inactivates both enveloped and non-enveloped viruses.

The sterile treated solution was transferred to an ion exchange columnloaded with Toyopearl CM-650C resin. The resin adsorbed gamma globulinand allowed all of the other proteins present in solution to flow out ineffluent from the column. After adding the solution to the column andadjusting the column flow to slowly drip out through the effluent end,effluent solution was measured at 280 nanometers to determine when allfree proteins and sterilants had been transported through the column.Afterward, the column was washed with a two times volume of purifiedwater to assure that the effluent has a very low measured opticaldensity at 280 nanometers.

A two percent solution of sodium chloride was then dispensed onto thetop of the column and allowed to percolate through the column. Gammaglobulin which was adsorbed by resin particles was freed to flow out ofthe column into a receiving vessel.

Collected effluent from the column with purified water (labeled asPurified Water) and effluent from the column with the two percentsolution (labeled as two percent NaCl) were tested electrophoreticallyto show the result of selected isolation and release of gamma globulinfrom the resin particles. Results of this step is summarized in TableVI, seen below:

TABLE VI Resulting percentage fractions Albumin Alpha 1 Alpha 2 Beta IgGPurified Water 26.0 2.7 15.2 57.0 nd* Two Pecent NaCl nd* nd* nd* 1.998.1 *nd = none detectedNote that more than 98% of the gamma globulin was isolated in the firstresin treatment. The value for beta globulin of 1.9% may be the resultof an application spot when applying solution to gel. The two percentsodium chloride solution contained the gamma globulin (IgG) and,perhaps, with larger pools of plasma, may contain some IgA and IgMglobulins which should be removed.

The two percent sodium chloride solution was therefore diafiltered toremove the sodium chloride for a next column treatment. Diafiltrationwas again performed by passing the solution though a Pellicon unitwhereby the salt was removed, yielding a final product which had aconductivity of 400 to 800 microSeimens/centimeter (μS/cm). Note that itlikely takes about six volumes of deionized purified water to diafilterthe two percent solution.

As a final step, a column was filled with Toyopearl 560-C resin and thedesalted solution was added to the top of the column and allowed toslowly percolate through the column. In this column, IgG flowed rightthrough the resin and all other proteins attached to the resin (e.g.,IgA and IgM) to yield a final effluent from the column (solution 172)that was 100% IgG in an aqueous base. The effluent tested is seen inTable VII below:

TABLE VII Percentage content of final solution Albumin Alpha 1 Alpha 2Beta IgG Solution 172 nd* nd* nd* nd* 100 *nd = none detected

Following the pathway of Charts 10- 1 and 10-2, other experiments havebeen performed using various salts in fractionating fresh frozen plasma.Steps 52, 54 and 64 were repeated using the various salts to completethe fractionation procedure in the two stages as outlined supra. As seenin Table VII, below, and consistent with the method disclosed for sodiumcitrate, first steps 52 and 54 comprised mixing a set volume of plasmawith a first predetermined volume of concentrated salt solution, wherepossible. Where it was not possible to achieve a necessary concentrationof salt by dissolving salt in water, dry salts were simply added toachieve the desired concentration, by weight.

The first predetermined volume of salt solution was twelve percent byweight. Reaction mixing followed at two to eight degrees Centigrade forone hour. After reaction mixing, the resulting mixture was spun down attwo to eight degrees Centigrade at 4500 rpm for one hour. A firstsupernatant fluid 60 overlying a paste precipitant 62 was gently pouredoff and collected. Note that the first supernatant fluid 60 contained atwelve percent solution, by weight, of the salt used. Generally, theresulting paste precipitant 62 was diluted with a percentage by weightof deionized water (usually four times the weight of collectedprecipitant 62) and stored at two to eight degrees overnight for furtheranalysis.

Per step 64, volume of supernatant 60 was measured and an amount ofconcentrated salt solution was added to raise the concentration of saltto a level of twenty-two percent, by weight. The resulting compositionwas mixed for one hour at two to eight degrees Centigrade and thenrefrigerated overnight. The following morning, the composition was mixedagain briefly for about five minutes and then centrifuged for one hourat 4500 rpm at two to eight degrees Centigrade. Any supernatant,supernatant 72, was poured off and saved for any desired furtherprocessing. Paste 70, the target of this procedure, after separationfrom supernatant 72, was weighed and diluted (redissolved per step 90,by adding an amount of deionized water calculated to be about four timesthe weight of paste 70).

The above disclosed procedure was repeated for both organic andinorganic salts in various combinations. Table VIII, shown below,provides results of the procedure as determined by serum electrophoresisperformed using the Beckman-Coulter system, Results of theelectrophoresis were scanned with the Beckman-Coulter scanner and arepresented as the percentage of protein at each of five levels, name asalbumin, alpha-1, alpha-2, beta and IgG.. The total for each scan, bythe Beckman algorithm, approximates 100% when summed

TABLE VIII Serum Electrophoresis Results of Salt Fractionations -showing % of each fraction Expt. Stage 1 Stage 2 Num 12% 22% AlbuminAlpha 1 Alpha 2 Beta IgG 1 Ammo- Ammo- 18.1 1.9 25.6 24.6 29.9 nium niumsulfate sulfate 2 Sodium Sodium 19.4 1.4 10.1 35.4 33.2 citrate citrate3 Ammo- Sodium 17.0 1.6 9.6 26.4 45.5 nium citrate sulfate 4 SodiumAmmo- 17.9 1.6 12.6 24.7 43.2 Citrate nium sulfate

There was no IgG found in the paste 62 nor in the supernatant 72. All ofthe IgG is found in paste 70. One should not be confused because valuesare higher for IgG for mixed organic (sodium citrate) and inorganic(ammonium sulfate) salts. Such is a result of a differential extractionof other proteins from the plasma. Some proteins have differentprecipitation patterns with different salts. The important result isthat substantially all of the IgG is found in the paste 70.

Results of a Procedure Performed via the Instant Inventive Method UsingEthanol

Surprisingly, it has been found that ethanol may be used within thescope of the instant invention as a fractionation compound. It should benoted that this use of ethanol is markedly distant from contemporary andhistorical methods employing ethanol in blood fractionation.

The method was tested and recorded in experimental laboratory notebooks,dated Dec. 8, 2005, using one frozen FFP-plasma bottle to provide acomparison with experiments disclosed supra. In this experiment, ethanolwas used in concentrations measured by percentage in the same mannerdisclosed for salts. Thus, a ninety-five percent ethanol solution wasdiluted to prepare a fifty percent ethanol solution (one hundred andfive milliliters of ninety-five was added to ninety-five milliliters ofdeionized water). So, prepared, the solution was placed in an ice bathto lower the temperature to five degrees Centigrade.

Per step 54, sixty-three milliliters of the fifty percent ethanolsolution was added to two hundred milliliters of FFP-plasma (from thespare) which was kept at five degrees Centigrade. The resulting solution(i.e. twelve percent ethanol) was stirred for one hour in an ice bath(at two to eight degrees Centigrade). After stirring, per step 56, thesolution was spun down for one hour using a Beckman J-6 centrifuge (4500rpm), while the temperature was maintained at two to eight degreesCentigrade. The supernatant 60 was poured off and the so separated paste62 was measured.

The volume of supernatant 60 was 260 milliliters. Weight of paste 62 was3.823 grams. Paste 62 was rehydrated with water (a four times volume,i.e. 15.3 milliliters and mixed well. Paste 62 showed a typicalclot-like precipitate. Final diluted volume was 18 milliliters.

Supernatant 60 was displaced into an ice bath. Additional fifty percentethanol (ninety-three milliliters) was added to supernatant 60 toincrease ethanol concentration to twenty-two percent, by weight (step64). This resulting solution was stirred for one hour at two to eightdegrees Centigrade, and then stored overnight in a refrigerator (at twoto eight degrees Centigrade).

The stored solution was stirred for about five minutes when taken fromthe refrigerator, then displaced into a centrifuge bottle and spun at4500 rpm for one hour at two to eight degrees Centigrade (per step 66).A supernatant 72 was poured off and a paste 70 was collected. It wasnoted that paste 70 was bright yellow in color. (Most commonly, paste 70is a gray-white color 31 when produced using salts). Though not knownexactly, it was suspected that the yellow color was due to billirubinand other chromogens being extracted from the plasma by the ethanol.Library samples were made at each fractionation step.

Supernatant volume was measured (336 milliliters) and stored at fivedegrees Centigrade. Paste 70 weighed 8.583 grams. Paste 70 wasrehydrated via a four times volume of deionized water (34.3milliliters), mixed well and stored overnight at two to eight degrees.It is interesting to note the unexpectedly high amount of IgG in paste70 when solvated.

Measured results are summarized in Tables IX, X and XI, below:

TABLE IX Serum Electrophoresis: Showing percent of each fraction.Albumin Alpha-1 Alpha-2 Beta IgG Paste 62 39.2 2.2 15.4 33.4  9.7Supernatant 72 73.7 9.1 8.3 8.9 — Paste 70 6.7 2.8 17.2 6.7 66.6

TABLE X Volume amounts of final fractionation. Sample Weight - gmVolume - ml FFP-plasma 200 Paste 62 3.823 18 Supernatant 72 336 Paste 708.583 40

TABLE XI Protein Results gm/dl - ml protein gm/liter - proteingm/liter - IgG FFP-Plasma 200 6.05 60.5 6.7 (11% - IgG) Paste 62 18 1.090.98 0.5 Supernatant 72 336 2.73 45.9 — Paste 70 40 4.75 9.5 6.3No electrophoresis was run on the initial base material. However, notethat there is about 6.7 grams of IgG per liter of plasma. If the IgG inpast 62 is added to IgG in paste 70, the total of 6.8 agrees well withthe anticipated 6.7 grams per liter.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiment is, therefore, to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are, therefore, intended to be embracedtherein.

1. A method of deriving a blood product from a blood based materialcomprising the steps of: (a) acquiring a predetermined quantity of theblood-based material for processing; (b) preparing the quantity ofblood-based material for processing; (c) selecting ethanol as a firstfractionation compound; (d) adding the first fractionation compound tothe quantity of blood-based material to yield a first separable productand a first residual product, wherein the first separable product iseuglobulin-depleted; (e) separating the first separable product from thefirst residual product to complete the first fractionation step; (f)selecting ethanol as a second fractionation compound; (g) adding asecond fractionation compound to the first separable product to yield asecond separable product and a second residual product, wherein thesecond separable product has a concentration of the second fractionationcompound that is greater than a concentration of the first fractionationcompound in the first separable product; (h) separating the secondseparable product from the second residual product to complete thesecond fractionation step; (i) preparing the second separable productfor diafiltration; and (j) diafiltering the second separable product toform a lower volume third product substantially free of the first andsecond fractionation compounds.
 2. The process for blood-based materialfractionation according to claim 1 wherein step (d) comprises adding aquantity of the first fractionation compound to produce a concentrationof 12% wt/wt.
 3. The process for blood-based material fractionationaccording to claim 1 wherein step (d) comprises adding a quantity of thefirst fractionation compound to produce a concentration within a rangeof 11-13% wt/wt.
 4. The process for blood-based material fractionationaccording to claim 1 wherein step (c) comprises selecting at least onesalt from a group of salts comprising sodium citrate, sodium acetate,sodium gluconate, ammonium sulfate, sodium chloride, sodium sulfate andammonium chloride to be used in place of ethanol as the firstfractionation compound.
 5. The process for blood-based materialfractionation according to claim 1 wherein step (g) comprises adding aquantity of the second fractionation compound to produce a concentrationof 22% wt/wt.
 6. The process for blood-based material fractionationaccording to claim 1 wherein step (g) comprises adding a quantity of thesecond fractionation compound to produce a concentration within a rangeof 21-23% wt/wt.
 7. The process for blood-based material fractionationaccording to claim 1 wherein step (f) comprises selecting at least onesalt from a group of salts comprising sodium citrate, sodium acetate,sodium gluconate, ammonium sulfate, sodium chloride, sodium sulfate andammonium chloride to be used in place of ethanol as the secondfractionation compound.